HIV-1 reverse transcriptase-pseudoknot RNA aptamer interaction has a binding affinity in the low picomolar range coupled with high specificity

Citation
O. Kensch et al., HIV-1 reverse transcriptase-pseudoknot RNA aptamer interaction has a binding affinity in the low picomolar range coupled with high specificity, J BIOL CHEM, 275(24), 2000, pp. 18271-18278
Citations number
52
Categorie Soggetti
Biochemistry & Biophysics
Journal title
JOURNAL OF BIOLOGICAL CHEMISTRY
ISSN journal
00219258 → ACNP
Volume
275
Issue
24
Year of publication
2000
Pages
18271 - 18278
Database
ISI
SICI code
0021-9258(20000616)275:24<18271:HRTRAI>2.0.ZU;2-F
Abstract
Systematic evolution of ligands by exponential enrichment (SELEX) is a powe rful method for the identification of small oligonucleotides that bind with high affinity and specificity to target proteins. Such DNAs/RNAs are a new class of potential chemotherapeutics that could block the enzymatic activi ty of pathologically relevant proteins. We have conducted a detailed bioche mical study of the interaction of human immunodeficiency virus I (HIV-1) re verse transcriptase (RT) with a SELEX-derived pseudoknot RNA aptamer. Elect ron paramagnetic resonance spectroscopy of site-directed spin-labeled RT mu tants revealed that this aptamer was selected for binding to the "closed" c onformation of the enzyme. Kinetic analysis showed that the RNA inhibitor b ound to HIV RT in a two-step process, with association rates similar to tho se described for model DNA/DNA and DNA/RNA substrates. However, the dissoci ation of the pseudoknot RNA from RT was dramatically slower than observed f or model substrates. Equilibrium binding studies revealed an extraordinaril y low K-d, of about 25 PM, for the enzyme-aptamer interaction, presumably a consequence of the slow off-rates. Additionally, this pseudoknot aptamer i s highly specific for HIV-1 RT, with the closely related HIV-2 enzyme showi ng a binding affinity close to 4 orders of magnitude lower.